1. Field of the Invention
The present invention relates to methods, compositions and kits for performing PCB immunoassays.
2. Discussion of the Background
Testing is an essential, and integral, component of all environmental protection and restoration activities. It is the rate limiting element that influences the time, cost, and overall efficiency of project management.
The management of toxic waste sites usually involves a progression through the stages of identification, characterization, remediation and monitoring, with testing being performed during each phase. Reference laboratory methods can effectively identify and quantify unknown compounds in a sample, but become relatively inefficient when used to rapidly locate contamination (i.e., mapping), and assist in remediation and monitoring activities. The complexity of laboratory protocols, and the proximity of the labs to the test site, delays the availability of information and increases the cost of data. The ultimate cost is in the time required by the field crews. Effective field screening methods can increase the efficiency of the clean-up process by providing an on-site, high-throughput, and cost-effective way to locate contamination and manage its remediation.
The Environmental Protection Agency (EPA) has long promoted and supported the concept of screening methods to supplement laboratory analysis and increase overall efficiency. The need for more effective methods has been recognized in the Superfund Amendments and Reauthorization Act of 1986 which specifies the development and evaluation of alternative time and cost-saving methods that will assist in the eventual remediation of the nations Superfund sites.
Effective field screening methods can increase the efficiency of site management and improve overall data quality when used to supplement the services of regional laboratories. The development of these methods, however, requires a technology that will be compatible with numerous compounds and matrixes and yet be simple, effective and rugged enough to be incorporated into a protocol for use in the field.
Screening methods need to provide fast, simple, cost-effective and reliable information when operated under field conditions. The reagents and equipment should be portable and stable at ambient conditions, and the claims relating to performance should accurately reflect anticipated field use. The methods should be able to rapidly provide an ample quantity of data, and the protocol should be simple to perform and safe to use. Performance characteristics relative to sensitivity, freedom from matrix interferences and cross-reacting compounds, and correlation to an acceptable reference method should be carefully evaluated. Developers must maintain high, and consistent, quality standards relative to the consistency of their manufacturing protocols, the adequacy of in-process and pre-release quality control methods, and the reliability of their product claims. A necessary characteristic of particular significance for screening methods, is that they exhibit a very low frequency of false negative results.
Screening methods detect contamination at specified concentrations. The concentration may relate to a hazardous threshold, a clean-up target, or a process-control parameter. The potential implications of false negative data far outweigh those of false positive results. The consequence of a false positive, while a costly problem that needs to be minimized, results in additional testing or treatment. False negative data, however, provides an erroneous perception of a clean site, and may have serious environmental and legal consequences. Safeguards that minimize the incidence of false negative results are imperative. Appropriate control over the frequency of false positive data needs to be established and maintained.
The field of immunochemistry,, and the development of immunoassay technology, has been evolving since the late 19th century. However, the majority of these methods have been developed for use by the medical community. These methods have achieved a reputation for reliability and cost-effectiveness. Literally hundreds of immunoassays have been developed for such applications as drug testing, Therapeutic Drug Monitoring (e.g. digitalis derivatives, anti-asthma formulations, anti-epileptic regents, antibiotics), pregnancy testing, hormone testing (e.g., thyroxine, thyroid stimulating hormone), tests for pathological markers (e.g. lactic dehydrogenase isozymes, creatine kinase isozymes), tests for acute phase proteins (e.g., carcinoembryonic antigen, alpha fetoprotein) and tests for tumor marker proteins.
Environmental applications have been explored for the better part of a decade and a number of immunoassay methods have been developed. Most have been used for the detection of herbicides and pesticides in aqueous matrixes. The application of immunoassay technology to the testing of solid waste, complex matrixes, and highly lipophilic compounds, has provided unique challenges for the chemist. The feasibility of developing such methods, however, has been demonstrated with immunoassays for single compounds such as Dioxin (see, for example, Vanderlaan et al, Environmental Toxicology and Chemistry, 7:859-870, 1988; and Stanker et al, Toxicology, 45:229-243, 1987).
The history of immunoassay technology can be traced to 1900 when Karl Landsteiner described the A, B and Zero (0) blood types after observing the agglutination reaction (i.e., aggregation) that resulted when he mixed the erythrocytes and serum for several of his co-workers on a slide. His observation became the basis for present day blood typing methods. Landsteiner remained a dominant figure in immunology for the next 40 years performing numerous experiments that demonstrated the extraordinary specificity of the antibody binding reaction. He introduced the term "hapten" to define compounds that are unable to directly stimulate antibody production when injected into an animal, but are capable of binding to an antibody if they are produced by an alternate means. Most environmental chemicals are haptens, and although potentially toxic, will not stimulate the immune system to respond.
For 50 years following Landsteiner's discovery, immunoassay technology continued to rely upon the binding and cross-linking ability of an antibody to cause agglutination, cell lysis, and protein flocculation reactions. These methods were relatively insensitive when compared to the immunoassay methods of today, and better suited to the analysis of larger compounds and organisms (e.g. bacteria, proteins). A major advance occurred in the 1950's when Drs. Berson and Yalow, while investigating the metabolism of radio-labelled insulin administered to diabetic patients, observed the production of anti-insulin antibodies in the serum of these patients (see Principles of Competitive Protein Binding Assays, Second Ed., Odell, W. D. and Franchismont, P. (Eds.); Wiley and Sons, New York). They described a radioimmunoassay (RIA) method in 1959 that used anti-insulin antibody molecules and radio-labelled insulin in a highly sensitive procedure to quantify insulin levels in the serum. The RIA method used a competitive antibody binding reaction, where radio-labelled insulin and sample insulin compete for a limited number of antibody binding sites. In 1977, Rosalyn Yalow was awarded the Nobel Prize in Medicine for her work on the development of the radioimmunoassay method for peptide hormones (see Basic and Clinical Immunology, 7th Ed. Stites, D. P. and Ten, A. I. editors; Appleton and Lange, Conn., 1991). RIA rapidly became a universally accepted method that demonstrated exceptional specificity, sensitivity, and simplicity.
A simpler, safer, and more convenient immunoassay was reported in 1971, when two independent research teams, Engvall and Perlmann, and Van Weeman and Shuurs, simultaneously disclosed a competitive immunoassay method that used an enzyme-labelled conjugate instead of a radio-labelled-conjugate to produce a test that generated a visible end-point signal (see Engvall et al, Immunochem. 8:871-874, 1971 and Van Weeman et al, FEBS Letters, 15:232-236, 1971). The new ELISA (i.e., enzyme linked immunosorbent assay) method eliminated the problems associated with the safety, disposal and detection of radioactive reagents. The method offered long term stability, the opportunity to generate quantifiable data using instruments commonly available in most laboratories, and a mechanism to develop separation-free (homogeneous) procedures and simple qualitative screening tests.
Current immunoassay technology benefits from the diversity of detection systems developed that use enzyme-catalyzed chromogenic reactions, radionuclides, chemiluminescence, fluorescence, fluorescence polarization and a variety of potentiometric and optical biosensor techniques. Improvements in the sensitivity achieved has necessitated the generation of new descriptive nomenclature for methods that can now detect "zeptomolar" (10.sup.-21, 600 molecules) concentrations.
Immunoassay methods combine the specific binding characteristics of an antibody molecule with a read-out system that is used to detect and quantify compounds. Antibodies are binding proteins that are produced by the immune system of vertebrates in response to substances that are perceived to be foreign.
The physiological role of antibody, or immunoglobulin, molecules is to bind, and thereby label for destruction, the perceived foreign substance. Antibody molecules are synthesized by a subset of lymphocytes, termed B lymphocytes, that become activated to produce antibody after exposure to substances having prerequisite size, complexity and "foreignness" to the host organism. Antibodies are large, polymeric proteins (i.e. .gtoreq.1.5.times.10.sup.5 d), that can be classified into sub-populations on the basis of their sequence, size and number of subunits. Five major populations, or isotypes, exist carrying the designations of IgM, IgA, IgD, IgG and IgE, with immunoglobulin G (IgG) usually found in the highest concentration.
Immunochemical assays are reliable when used in the screening of soil for contamination and have been used commercially for the rapid analysis of a variety of compounds (see Nqo, in Enzyme-Mediated Immunoassay, eds., Debtor et al, Plenum Press: New York, 3 (1985); Odell, in Principles of Competitive Protein-binding Assays; eds., Odell et al, J. Wiley & Sons, New York, 1 (1971); Quantitative Enzyme Immunoassay; Blackwell Scientifid, Oxford (1978); Engvall, in Enzyme Immunoassay, eds., E. Ishikawa et al, Igaku-Shoin, New York, 1 (1981); Jaklitsch, in Enzyme-Mediated Immunoassay, eds., Debtor et al, Plenum Press, New York, 33 (1985)), and have been developed to detect a number of different compounds of environmental concern (see Immunochemical Methods for Environmental Analysis; ACS Symposium Series 442; Amer. Chem. Soc., Washington, DC (1990); Mapes et al, Bull. Environ. Contam. Toxicol. 49, in press (1992); Immunoassays for Trace Chemical Analysis; ACS Symposium Series 451, Amer. Chem. Soc., Washington, DC (1990); Harrison et al, in Biotechnology for Crop Production, Hedin et al, eds., ACS Symposium Series 379; Amer. Chem. Soc.: Washington, DC, 316 (1988); Hammock et al, in Recent Advances in Pesticide Analytical Methodology; Harvey et: al, eds., ACS Symposium Series 136; Amer. Chem. Soc.: Washington, DC, 321 (1980); Van Emon et al, in Analytical Methods for Pesticide and Plant Growth Regulators Vol. XXII, 217 (1989); Albro et al, Tox. & Appl. Pharm. 50:137 (1979); Blewett et al, Bull. Environ. Contam. Toxicol. 45:120 (1990); Bushway et al, Bull. Environ. Contam. Toxicol. 40:647 (1988); Fleeker et al, in Immunoassays for Monitoring Human Exposure to Toxic Chemicals; Vanderlaan et al, eds., ACS Symposium Series #451 (1991); Gee et al, J. Agric. Food Chem. 36:863 (1988); Goh et al, Bull. Environ. Contam. Toxicol. 46:30 (1991); Harrison et al, Agric. Food Chem. 37:958 (1989); Jung et al, J. Agric. Food Chem. 37:1183 (1989); Jung et al, Pesticide Science 26:303 (1989); Thurman et al, Anal. Chem. 62:2043 (1990)).
One of the most serious problems in environmental contamination is the presence of polychlorinated biphenyls (PCBs). PCBs, as commercially available, exist as mixtures of PCB congenors containing various mixtures of 209 different isomeric forms. These mixtures were distributed commercially under the commercial name AROCLOR. A number assigned to the AROCLOR designation indicates average percent chlorination of the PCB congenors in the product. Thus, AROCLOR 1260 contains PCBs with an average chlorination of 60%, AROCLOR 1254 has an average chlorination of 54%, AROCLOR 1248 has an average chlorination of 48%, AROCLOR 1242 has an average chlorination of 42%, and AROCLOR 1232 has an average chlorination of 32%. The only one of the important toxic AROCLORs which does not follow the above rule is AROCLOR 1016, which has an average chlorination of 41%. Toxicological data has indicated that the highly chlorinated PCBs are the most toxic to human health. The most frequently encountered toxic AROCLORs are AROCLOR 1260, AROCLOR 1254, AROCLOR 1016, AROCLOR 1232, AROCLOR 1242 and AROCLOR 1248. However, since the composition of PCB products varies from individual product to individual product, from manufacturer to manufacturer within the same product, and even from lot to lot within the same product, immunoassay test development is difficult.
Mattingly et al, U.S. Pat. No. 5,145,790, disclose an immunoassay based method for detecting polychlorinated biphenyls. However, the Mattingly et al method requires the use of a combination of antibodies in order to detect both the highly chlorinated biphenyls, such as AROCLORs 1260 and 1254, as well as the lower chlorinated biphenyls, including AROCLORs 1016, 1221, 1232, 1242, and 1248. Thus, in order to use Mattingly et al in a screening program at a contamination site, one would be required to either test each sample multiple times with different antibodies, or to use mixtures of antibodies in a test.
Under the Toxic Substances Control Act, the U.S. Environmental Protection Agency (EPA) requires the cleanup of all spills and discharges where the spilled material contains more than 50 ppm of PCBS. Contaminated surfaces are required to be cleaned to 10 .mu.g/100 cm.sup.2 or 100 .mu.g/100 cm.sup.2, depending on the nature of the surface. To determine (1) if a cleanup is necessary and (2) whether such a cleanup has been effective, the EPA requires that standard wipe tests be performed on the surface. In 40 CFR 761.123 the EPA defines a "standard wipe test":
. . "Standard wipe test" means, for spills of high concentration PCBs on solid surfaces, a cleanup to numerical surface standards and sampling by a standard wipe test to verify that the numerical standards have been met. This definition constitutes the minimum requirements for an appropriate wipe testing protocol. A standard size template (10 centimeters (cm).times.10 cm) will be used to delineate the area of cleanup; the wiping medium will be a gauze pad or glass wool of known size which has been saturated with hexane. It is very important that the wipe be performed very quickly after the hexane is exposed to air. EPA strongly recommends that the gauze (or glass wool) be prepared with hexane in the laboratory and that the wiping medium be stored in sealed glass vials until it is used for the wipe test. Further, EPA requires the collection and testing of field blanks and replicates.
In 40 CFR 761.130(e) the EPA also recommends a study by the Midwest Research Institute (MRI) which describes a standard wipe test in more detail. The report proposal leaves a considerable amount of latitude to the analyst as to how the sampling and analysis are to be performed ("Verification of PCB Spill Cleanup by Sampling and Analysis", pp. 41-42).
Thus, an immunoassay method is needed which will provide reliable, accurate and fast results in the field for a wide range of PCB contaminants in a single test using a single antibody, regardless of manufacturer, exact composition or matrix. Such an assay would increase the efficiency of environmental site management activities such as characterization (mapping), remediation monitoring, and regulatory compliance.
In addition, a standard PCB wipe test is needed which will provide reliable, accurate and fast field results in accordance with EPA guidelines.